Trafficking of apical transporters and enzymes to the enterocyte brush border is critical for the establishment of normal fluid, nutrient and electrolyte absorption from the gut lumen. Similarly, establishment of the normal compendium of proteins at the intercellular tight and adherens junctions determines the integrity of epithelial barrier function. Aberrations in either apical trafficking or junctional integrity lead to enteric pathologies including diarrhea, constipation and intestinal barrier dysfunction. Investigations over the past decade have established that components of the Rab11a and Rab8a-dependent membrane recycling systems regulate trafficking of apical membrane and junctional proteins required for the maintenance of proper apical polarity. Loss of functional Myosin Vb (MYO5B) leads to severe diarrhea due to loss of apical transporters secondary to deficits in Rab11a and Rab8a-dependent apical trafficking as observed in neonates with Microvillus Inclusion Disease (MVID). At the same time, the Rab11a-interacting proteins, Rab11-FIP1 and Rab11-FIP2, regulate junctional structure and function, in part through their phosphorylation by the critical polarity-related kinase MARK2/Par1b. All of these studies have led to the recognition that the coordinated regulation of apical polarity and trafficking by components of the apical recycling system lies at the heart of enterocyte physiology and pathophysiology. We have hypothesized that pathways through aspects of the recycling system provide for specificity of trafficking of particular cargoes to the apical membrane and the intercellular junctions. The apical recycling system represents a critical target for intervention in pathophysiologies that undermine apical membrane trafficking to the brush border and establishment of functional junctional integrity. To analyze this hypothesis, we will pursue two specific aims: First, we will determine the roles of elements of the apical recycling system in trafficking of ion transporters and enzymes to the enterocyte apical brush border. These studies will examine the fates of endogenous enterocyte apical proteins in human intestinal enteroids differentiated on two-dimensional monolayer cultures on permeable membranes. Second, we will examine the role of Rab11-FIP1B and its phosphorylation by MARK2/Par1b in the regulation of intestinal enterocyte junctional function. These studies will allow a greater understanding of how phosphorylation of Rab11-FIP1B regulates trafficking to and maintenance of junctions. Impact: These studies will identify components of the apical recycling system that may regulate apical trafficking of particular brush border and junctional cargoes. Definition of the multiple pathways for apical cargo trafficking to the brush border and junctions will provide insights into how disruption of specific pathways may alter physiology and how pathophysiological loss of trafficking may be restored through rerouting of proteins to alternative routes to the apical membrane.!